View source for Pyrosequencing

{{Short description|Method of DNA sequencing}}
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'''Pyrosequencing''' is a method of [[DNA sequencing]] (determining the order of [[nucleotides]] in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a [[DNA polymerase]]. Pyrosequencing relies on light detection based on a chain reaction when [[pyrophosphate]] is released, hence, the name given it.

==Principles==
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The principle of pyrosequencing was first described in 1993 by [[Pål Nyrén|P. Nyrén]], B. Pettersson, and [[Mathias Uhlén|M. Uhlen]].{{primary source inline|date=October 2025}}<!--ONE CANNOT ESTABLISH A DISCOVERY FROM THE PRIMARY SOURCES OF A SUGGESTED DISCOVERER. SECONDARY SOURCES ARE ABSOLUTELY REQUIRED.--><ref name = NyrenPetterssonUhlen1993>{{cite journal | author = Nyrén, P.; Pettersson, B. & Uhlen, M. | date = January 1993 | title = Solid Phase DNA Minisequencing by an Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay | journal = Analytical Biochemistry | volume = 208 | issue = 1 | pages = 171–175 | issn = 0003-2697 | url = https://www.sciencedirect.com/science/article/pii/S0003269783710249 | access-date = 28 October 2025 | url-access = subscription | doi = 10.1006/abio.1993.1024 | pmid = 8382019 }}{{primary source inline|date=October 2025}}</ref><!--Who was corresponding author? Also, no full names to appear until a source is presented that states them. Anal Bioch article only presents first initials.--> The technique combines [[solid phase sequencing]], and use of [[streptavidin]]-coated magnetic beads, a recombinant DNA polymerase lacking 3´-to-5´exonuclease activity (proof-reading), and luminescence detection of inorganic [[pyrophosphate]] using the firefly [[luciferase]] enzyme.{{primary source inline|date=October 2025}}<!--See note in all caps above.--><ref>{{cite journal | author = Uhlen, M. | date = 31 August 1989 | title = Magnetic Separation of DNA | journal = [[Nature (journal)|Nature (London)]] | volume = 340 | issue =  6236| pages = 733–734 | issn =  | url = https://www.nature.com/articles/340733a0#citeas | access-date = 28 October 2025 | url-access = subscription | doi = 10.1038/340733a0 | pmid = 2770876 | bibcode = 1989Natur.340..733U }}{{primary source inline|date=October 2025}}</ref><ref>{{cite journal | author = Nyrén, Pål & Lundin, Arne | date = December 1985 | title = Enzymatic Method for Continuous Monitoring of Inorganic Pyrophosphate Synthesis | journal = Analytical Biochemistry | volume = 151 | issue = 2 | pages = 504–509 | issn = 0003-2697 | url = https://dx.doi.org/10.1016/0003-2697%2885%2990211-8 | access-date = 28 October 2025 | url-access = subscription | doi = 10.1016/0003-2697(85)90211-8 | pmid = 3006540 }}{{primary source inline|date=October 2025}}</ref>{{what|date=October 2025}}

Specifically, a solution of three [[enzyme]]s—[[DNA polymerase]], [[Sulfate adenylyltransferase|ATP sulfurylase]], and firefly [[luciferase]]—and a deoxyribo[[nucleoside triphosphate]] (dNTP) are added to single stranded DNA to be sequenced, and the incorporation of nucleotide is followed, measuring the light emitted as a consequence of inorganic pyrophosphate production.{{cn|date=October 2025}} The intensity of the light determines if 0, 1, or more nucleotides have been incorporated, thus showing how many complementary nucleotides are present on the template strand.{{cn|date=October 2025}} The nucleotide mixture is removed before a next nucleotide mixture is added, and the process is repeated for each of the four nucleotides, until the DNA sequence of the single stranded template is determined.{{cn|date=October 2025}}

A second solution-based method for pyrosequencing was described in 1998 by [[Mostafa Ronaghi]], [Mathias Uhlen],<ref>{{cite web | title=School of Engineering Sciences in Chemistry, Biotechnology and Health | url=https://www.kth.se/en/bio/research/proteomics/proteomics-researchers/mathias-uhlen-1.67763 }}</ref> and [[Pål Nyrén|Pål Nyren]].{{primary source inline|date=October 2025}}<!--See note in all caps above.--><ref>{{Cite journal |last1=Ronaghi |first1=Mostafa |last2=Uhlén |first2=Mathias |last3=Nyrén |first3=Pål |date=1998-07-17 |title=A Sequencing Method Based on Real-Time Pyrophosphate |url=https://www.science.org/doi/abs/10.1126/science.281.5375.363 |journal=Science |volume=281 |issue=5375 |pages=363–365 |language=en |doi=10.1126/science.281.5375.363|pmid=9705713 |s2cid=26331871 |url-access=subscription }}{{primary source inline|date=October 2025}}</ref> In this alternative method, an additional enzyme, [[apyrase]], is introduced to remove nucleotides that are not incorporated by the DNA polymerase.{{cn|date=October 2025}} This enables the enzyme mixture— [[DNA polymerase]], [[luciferase]], and [[apyrase]]—to be added when sequencing is initiated, and kept in the reaction solution throughout the procedure (thus enabling easier automation).{{cn|date=October 2025}} An automated instrument based on this principle was introduced to the market the following year by the company Pyrosequencing.{{cn|date=October 2025}}

A third variant, a microfluidic pyrosequencing method, was described in 2005 by an industrial research team led by [[Jonathan Rothberg]], at the company [[454 Life Sciences]].{{primary source inline|date=October 2025}}<!--See note in all caps above.--><ref>{{cite journal | author = Margulies, M.; Egholm, M.; Altman, W.E.; Attiya, S.; Bader, J.S.; Bemben, L.A.; Berka, .; Braverman, M.S.; Chen, Y.-J.; Chen, Z.; Dewell, S.B.; Du, L.; Fierro, J.M.; Gomes, X.V.; Godwin, B.C.; He, W.; Helgesen, S.; Ho, C.H.; Irzyk, G.P.; Jando, S.C.; Alenquer, M.L.I.; Jarvie, T.P.; Jirage, K.B.; Kim, J.-B.; Knight, J.R.; Lanza, J.R.; Leamon, J.H.; Lefkowitz, S.M.; Lei, M.; Li, J.; Lohman, K.L.; Lu, H.; Makhijani, V.B.; McDade, K.E.; McKenna, M.P.; Myers, E.W.; Nickerson, E.; Nobile, J.R.; Plant, R.; Puc, B.P.; Ronan, M.T.; Roth, G.T.; Sarkis, G.J.; Simons, J.F.; Simpson, J.W.; Srinivasan, M.; Tartaro, K.R.; Tomasz, A.; Vogt, K.A.; Volkmer, G.A.; Wang, S.H.; Wang, Y.; Weiner, M.P.; Yu, P.; Begley, R.F. & Rothberg, J.M. | author-mask =  | date = 31 July 2005 | title = Genome Sequencing in Microfabricated High-Density Picolitre Reactors | journal = [[Nature (journal)|Nature (London)]] | volume = 437 | issue =  7057| pages = 376–380 | issn =  | doi = 10.1038/nature03959 | pmid = 16056220| pmc = 1464427| bibcode = 2005Natur.437..376M}}{{primary source inline|date=October 2025}}</ref> This alternative approach for pyrosequencing was based on the original principle of attaching the DNA to be sequenced to a solid support; Rothberg and co-workers demonstrated that sequencing could be performed in a highly parallel manner using a [[microfabrication]] and [[microarray]]s.{{cn|date=October 2025}} This allowed high-throughput DNA sequencing, and an automated instrument was introduced to the market.{{cn|date=October 2025}} This first next generation sequencing instrument initiated a new era in [[genomics]] research,{{says who|date=October 2025}} and to rapidly falling prices for [[DNA sequencing]],{{says who|date=October 2025}} allowing affordable [[whole genome sequencing]].{{cn|date=October 2025}}

==Procedure ==
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[[File:How Pyrosequencing Works.svg|alt=How Pyrosequencing Works|thumb|679x679px|The chart shows how pyrosequencing works.]]
"Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of [[DNA polymerase]] (a DNA synthesizing enzyme) with another [[chemiluminescence|chemoluminescent]] [[enzyme]].  Essentially, the method allows sequencing a single strand of [[DNA]] by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T [[nucleotides]] are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.<ref name=Qiagen>{{cite web| author = Qiagen Staff | date = 4 August 2017 | title=Pyrosequencing Technology and Platform Overview | work = Qiagen Knowledge & Support/Knowledge Hub/Technology & Research/Pyrosequencing Resource Center (Qiagen.com) | url=https://www.qiagen.com/us/resources/technologies/pyrosequencing-resource-center/technology-overview/| access-date=4 August 2017}}{{better source|date=October 2025}}</ref>{{better source|date=October 2025}}<!--This commercial source will lean in the direction of the product they are selling, and so is a poorer source than a published review or other secondary source.-->{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.-->

For the solution-based version of pyrosequencing, the single-strand DNA ([[ssDNA#ssDNA|ssDNA]]) template is hybridized to a sequencing [[Primer (molecular biology)|primer]] and incubated with the enzymes [[DNA polymerase]], [[ATP sulfurylase]], [[luciferase]] and [[apyrase]], and with the substrates [[adenosine 5´ phosphosulfate]] (APS) and [[luciferin]].<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.-->

# The addition of one of the four [[deoxynucleotide triphosphate]]s initiates the second step; [[dNTP]]s)—dATPαS, which is not a substrate for a luciferase, is added instead of dATP to avoid noise. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases [[pyrophosphate]] (PPi).<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.-->

# ATP sulfurylase converts PPi to [[Adenosine triphosphate|ATP]] in the presence of adenosine 5´ phosphosulfate. This ATP acts as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount.{{what|date=October 2025}} The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a program.<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.-->

# Unincorporated nucleotides and ATP are degraded by the [[apyrase]], and the reaction can restart with another nucleotide.<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.-->

The process can be represented by the following equations:
* PPi + APS → ATP + Sulfate (catalyzed by ATP-sulfurylase);
* ATP + luciferin + O2 → AMP + PPi + oxyluciferin + {{CO2}} + hv (catalyzed by luciferase);
where PPi is pyrophosphate, APS is adenosine 5-phosphosulfate, ATP is adenosine triphosphate, O2 is di[[oxygen]], AMP is adenosine monophosphate, {{CO2}} is carbon dioxide, and hv is light.<ref name=Qiagen/>{{verification needed|date=October 2025}}<!--We need to verify that all content preceding this in the paragraph, is from this source.-->

==Limitations==
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Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with [[DNA sequencing#Chain-termination methods|chain termination]] methods (e.g. Sanger sequencing).{{cn|date=October 2025}}  This can make the process of [[genome assembly]] more difficult, particularly for sequences containing a large amount of [[repetitive DNA]].{{cn|date=October 2025}} Also, lack of a proof-reading activity{{what|date=October 2025}} limits accuracy of this method.{{cn|date=October 2025}}

==Commercialization==
Pyrosequencing AB, a company based in [[Uppsala, Sweden]], was founded with [[venture capital]] provided by [[HealthCap]] in order to commercialize machinery and reagents for sequencing short stretches of DNA using the pyrosequencing technique.<ref name=BiotageHistory/>{{verification needed|date=October 2025}} Pyrosequencing AB was listed on the [[Stockholm Stock Exchange]] in 1999.<ref name=BiotageHistory/>{{verification needed|date=October 2025}} When Pyrosequencing AB acquired Biotage LLC, a U.S.-based company, and other companies, in 2003, the company was renamed Biotage AB.<ref name=BiotageHistory>{{Cite web |author=Biotage Staff |title=Biotage History |url=https://www.biotage.com/biotage-history |access-date=2022-09-19 |work=Biotage.com |language=en}}</ref> The pyrosequencing and other biomedical units of Biotage AB were sold to [[Qiagen]] in 2008.<ref name=BiotageHistory/>{{verification needed|date=October 2025}}  The pyrosequencing technology was licensed to [[454 Life Sciences]].{{when|date=October 2025}}{{cn|date=October 2025}} 454 developed an array-based pyrosequencing technology that emerged as a platform for [[DNA sequencing#High-throughput sequencing|large-scale DNA sequencing]], including [[genome project|genome sequencing]] and [[metagenomics]].{{cn|date=October 2025}}

[[Hoffmann-La Roche|Roche]] acquired [[454 Life Sciences]],{{when|date=October 2025}}{{cn|date=October 2025}} and announced the discontinuation of the 454 sequencing platform in 2013.<ref>{{cite news|author =Hollmer, Mark| date=October 17, 2013 | title=Roche to Close 454 Life Sciences as it Reduces Gene Sequencing Focus | work = [[Fierce Biotech]] | url=http://www.fiercebiotech.com/medical-devices/roche-to-close-454-life-sciences-as-it-reduces-gene-sequencing-focus| access-date = 28 October 2025}}</ref> The 454 sequencing platform was replaced, in part, by [[Illumina dye sequencing]], and by Applied Biosystems sequencing products.{{cn|date=October 2025}}

==Further reading==
* {{cite journal| author= Harrington, C.T.; Lin, E.I.; Olson, M.T. & Eshleman, J.R. | date = September 1, 2013 | title = Fundamentals of Pyrosequencing | journal = Arch. Pathol. Lab. Med. | volume = 137 | issue = 9 | pages = 1296–1303 | url = https://meridian.allenpress.com/aplm/article/137/9/1296/193658/Fundamentals-of-Pyrosequencing | url-access =subscription | access-date = 29 October 2025 | doi = 10.5858/arpa.2012-0463-RA | pmid = 23991743 }} For the corresponding PDF document, see [https://allen.silverchair-cdn.com/allen/content_public/journal/aplm/137/9/10.5858_arpa.2012-0463-ra/2/arpa_2012-0463-ra.pdf?Expires=1764740125&Signature=G9NXpBjO~irAudtDoIXycecX~4lHevw~xMEYSc~6DZTDT~ctj6sICV4afDIAu6Nkza6Hfky5~5hE3hTuYpnUenRfUj2VLfIz5BQx17FoIp3sI4CoxjzI16ACtBq~cLi4A48-CAuPrpsJY5-4bWolqNHQpc5GCTdzLl1VkYknDcc4TlRYBt89l7XmfJ-bFIJak0S2T8M7L-Dth19D0z8UPIPbIo4nuaPergMq2Gf0q0Rn9GZlLLaeePPuiEgcAISUYX3PezwNrAUGXmeEnuBSAmUAhlH82h0zNcibUsdlUpSqpKQRjMg5beyISWDK8zsZ~xrSMgjraiZ9BxZZHLNkNA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA this link].

* {{cite journal|author=Metzker M. |title=Emerging Technologies in DNA Sequencing|journal=Genome Research |date=2005| pmid=16339375|volume=15|pages=1767–76|doi=10.1101/gr.3770505|issue=12|doi-access=free}}

==References==
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[[Category:Biotechnology]]
[[Category:DNA sequencing methods]]
[[Category:Life sciences industry]]
[[Category:Molecular biology]]